The accurate flow of genetic information from DNA to RNA to protein is essential for all living organisms. An astonishing array of quality-assurance mechanisms have evolved to ensure that high degree of fidelity is maintained at every stage of this process. One of the most fascinating quality control mechanisms involves tmRNA, also known as SsrA or 10Sa RNA. tmRNA is a versatile and highly conserved bacterial molecule endowed with the combined structural and functional properties of both a tRNA and an mRNA. Our previous studies have shown that all known activities of tmRNA require SmpB, a small protein that binds tmRNA specifically and with high affinity to promote its association with stalled ribosomes. The SmpB-tmRNA system orchestrates three key biological functions: 1) recognition and rescue of ribosomes stalled on aberrant mRNAs, 2) disposal of the causative defective mRNAs, and 3) addition of a degradation tag to the incomplete protein fragments for directed proteolysis. Although not essential in E. coli, tmRNA activity is essential for bacterial survival under adverse conditions and for virulence in some, and perhaps all, pathogenic bacteria. Recent evidence from our laboratory suggests that in addition to its quality control function the tmRNA system might also play a key regulatory role in certain physiological pathways. Moreover, because the SmpB and tmRNA are found only in prokaryotes, involves novel RNA and protein factors, and is essential for the survival of pathogenic bacteria, a deeper mechanistic understanding of this system might allow the design of highly specific new anti-bacterial agents. The molecular basis for the formation of the SmpB-tmRNA complex and the subsequent recognition of stalled ribosomes are not well understood. The objective of this research program is to use a combination of molecular genetics, protein biochemistry, bioinformatics, and structural approaches to elucidate the mechanism of action of the SmpB-tmRNA quality control system. The emphasis is on the molecular characterization of how SmpB-tmRNA complex recognizes stalled ribosomes and promotes the detection and selective decay of the causative defective mRNA by the 3'-5' exonuclease RNase R. Specifically, through these studies we wish to understand the biochemical and structural basis for the interactions of SmpB and RNase R with tmRNA and the ribosome; i.e. what amino acid residues are involved, what base specific contacts are made, what structural features contribute to the formation of the tmRNA-associated SmpB and RNase R complexes and their interaction with stalled ribosome. PUBLIC HEALTH RELEVANCE: As currently available antibiotics lose their effectiveness the need for new counter measure becomes ever more urgent. The genetic, biochemical, and structural studies outlined here offer the opportunity to gain novel insights into and a deeper mechanistic understanding of a unique bacterial surveillance system mediated by the versatile tmRNA and its essential protein partner, SmpB. A thorough understanding of this extraordinary bacterial system, essential for survival and virulence of many pathogenic bacteria, should pave the way for development of knowledge-based new anti-infective agents that exclusively target pathogenic microorganisms. Ultimately, these insights will have implications for a better understanding of a variety of cellular processes, including control of gene expression, synthesis and degradation of proteins, and the targeted decay defective mRNAs.